Method for diagnosis of and following a bacterial vaginosis by molecular quantification

Abstract

The present invention relates to a method for in vitro diagnosis and follow-up of vaginal bacterial flora status in relation to the presence of bacterial vaginosis, and for monitoring its treatment where applicable, wherein the presence of bacterial vaginosis or failure of on-going treatment is determined if the concentrations of specific sequences present in a single copy in the DNA of the bacteria Atopobium vaginae and Gardnerella vaginalis in the DNA extracted from a vaginal discharge specimen from a patient are such that at least one of the following two conditions a) and b) are met: a) the concentration Ca of said DNA fragment of Atopobium vaginae is greater than or equal to 10 8 copies/mL, and b) the concentration Cg of said DNA fragment of Gardnerella vaginalis is greater than or equal to 10 9 copies/mL.

1 . A method for in vitro diagnosis and follow-up of the vaginal bacterial flora status in relation to the presence of bacterial vaginosis, and for monitoring its treatment where applicable, the method comprising:
 1) quantifying the concentrations of the bacteria  Atopobium vaginae  and  Gardnerella vaginalis  by
 determining the concentrations of-the specific sequences of said bacteria  Atopobium vaginae  and  Gardnerella vaginalis  present in a single copy in the DNA of said bacteria  Atopobium vaginae  and  Gardnerella vaginalis,  and of a specific sequence of a human gene present in all biological specimens containing human cells, in the DNA extracted from a vaginal discharge specimen from a patient, said specific sequences being less than 150 nucleotides in size, 
 enzymatically co-amplifying said specific sequences contained, on the one hand, in said DNA extracted from the specimen and, on the other hand, in samples of synthetic DNA fragments including each of said specific sequences of said bacteria and said specific sequence of a human gene present in all biological human cell specimens, said samples serving as calibration standards for quantifying the DNA, 
 detecting and quantifying said amplified fragments with the aid of probes labeled with sequences different from those of the amplification primers for each of said specific sequences of said bacteria  Atopobium vaginae  and  Gardnerella vaginalis  and said specific sequence of a human gene present in all biological human cell specimens, and 
   2) determining the presence of bacterial vaginosis or failure of the on-going treatment if the concentrations of DNA fragments of said two specific sequences of the bacteria  Atopobium vaginae  and  Gardnerella vaginalis  in a specimen of the patient's vaginal discharge containing at least 104 human cells/mL are such that at least one of the following two conditions a) and b) is met:
 a) the concentration Ca of said DNA fragment of  Atopobium vaginae  is greater than or equal to 108 copies/mL, and 
 b) the concentration Cg of said DNA fragment of  Gardnerella vaginalis  is greater than or equal to 109 copies/mL. 
   
     
     
         2 . The method according to  claim 1 , wherein:
 a) in step 1), the  Lactobacillus  sp. including at least the bacteria  Lactobacillus acidophilus, Lactobacillus crispatus, Lactobacillus jenseneii, Lactobacillus gasseri  and  Lactobacillus iners  are additionally quantified
 by additionally determining the concentration of a specific sequence of said  Lactobacillus  sp. bacteria, said specific sequence of  Lactobacillus  sp. being present in a single copy in the DNA of said  Lactobacillus  sp. bacteria, and being less than 150 nucleotides in size, 
 by additionally ezymatically co-amplifying said specific sequence of  Lactobacillus  sp. contained, on the one hand, in said DNA extracted from the specimen and, on the other hand, in a specimen of synthetic DNA fragments including, additionally, said specific sequence of said  Lactobacillus  sp. bacteria, with said synthetic DNA fragment including said specific sequence of said  Lactobacillus  sp. bacteria serving as a quantification reference standard. 
 detection and quantification of said amplified fragments being accomplished with the aid of labeled probes with sequences different from those of the amplification primers for each of said specific sequences of said  Atopobium vaginae, Gardnerella vaginalis,  and  Lactobacillus  sp. bacteria and said specific sequence of a human gene present in any biological specimen containing human cells, and 
   b) in step 2), bacterial vaginosis is determined if, additionally, the Cl concentration of a specific DNA fragment of said  Lactobacillus  sp. bacteria is less than or equal to 108 copies/mL.   
     
     
         3 . The method according to  claim 2 , wherein the presence of bacterial vaginosis or failure of the on-going treatment is determined if said concentrations are such that the following three conditions are met:
 a—Concentration Ca of said DNA fragment of a specific sequence of  Atopobium vaginae  is greater than or equal to 108 copies/mL,   b—Concentration Cg of said DNA fragment of a specific sequence of  Gardnerella vaginalis  is greater than or equal to 109 copies/mL, and   c—Concentration Cl of said DNA fragment of a specific sequence of  Lactobacillus  sp. is less than or equal to 107 copies/mL.   
     
     
         4 . The method according to  claim 1 , wherein real-time PCR amplification and quantification reactions are performed by using hydrolysis probes specific to each of said specific sequences of said bacteria and specific sequence of a human gene present in any biological specimen containing human cells, in the specimen to be tested. 
     
     
         5 . The method according to  claim 4 , wherein said specific sequences are 70 to 150 nucleotides in size. 
     
     
         6 . The method according to  claim 1 , wherein a large synthetic DNA fragment serving as a reference standard for DNA quantification is used, said large synthetic DNA fragment grouping said specific sequences of each of said bacteria whose concentrations are quantified, and said specific human DNA sequence of human cells. 
     
     
         7 . The method according to  claim 1 , wherein said specific sequences of said bacteria include:
 for the  Atopobium vaginae  bacterium: the fragment of positions 248 to 334 of the 16S ribosomal RNA gene, GenBank reference AY 738658.1,   for the  Gardnerella vaginalis  bacterium, the fragment of positions 981 to 1072 of the Cpn 60 gene of the chaperone protein 60 kDa, GenBank reference AF 240579.3, and   for the  Lactobacillus  sp. bacterium, a sequence common to the bacteria  Lactobacillus crispatus, Lactobacillus jenseneii, Lactobacillus gasseri, Lactobacillus iners  and  Lactobacillus acidophilus  in the tuf gene coding for the elongation factor at positions 253 to 343 of the gene with GenBank reference AY 562191.1.   
     
     
         8 . The method according to  claim 7 , wherein said specific sequences of said bacteria are the following sequences, including probe sequences framed by primer sequences or their complementary sequences:
 the sequence as set forth in SEQ ID NO:2 for  Atopobium vaginae,      the sequence as set forth in SEQ ID NO:3 for  Gardnerella vaginalis,  and   the sequence as set forth in SEQ ID NO:4 for  Lactobacillus  sp.   
     
     
         9 . The method according to  claim 6 , wherein said large DNA fragment includes, as a specific sequence of human DNA in the test sample, a specific albumin sequence. 
     
     
         10 . The method according to  claim 9 , wherein said specific sequence of human DNA in the test specimen includes the fragment of positions 16283-16423 of exon 12 of the human albumin gene with GenBank reference M12523.1 with the sequence as set forth in SEQ ID NO:1 or the complementary sequence thereof. 
     
     
         11 . The method according to  claim 10 , wherein said specific sequence of the human DNA in the test specimen including the fragment of positions 16283-16423 of exon 12 of the human albumin gene GenBank reference M12523.1, is modified by insertion of a cleavage site outside the sequences corresponding to the primers and the probe sequence of the sequence as set forth in SEQ ID NO:17. 
     
     
         12 . The method according to  claim 1 , wherein sets of primers and probes are chosen where applicable from the following sequences or their complementary sequences:
 for  Atopobium vaginae      SEQ ID NOs:5-7 for the 5′ primer, the 3′ primer, and the probe, respectively;   for  Gardnerella vaginalis      SEQ ID NOs:8-10 for the 5′ primer, the 3′ primer, and the probe, respectively;   for  Lactobacillus  sp.   SEQ ID NOs:11-13 for the 5′ primer, the 3′ primer, and the probe, respectively; and   for human albumin   SEQ ID NOs:14-16 for the 5′ primer, the 3′ primer, and the probe, respectively.   
     
     
         13 . The method according to  claim 6 , wherein said large synthetic DNA fragment constituting the DNA of the standard reference specimen for quantification is inserted into a plasmid. 
     
     
         14 . Diagnostic kit used for implementing a method according to  claim 1  comprising:
 samples of standard reference DNA including said specific sequences of each of said bacteria, as well as   said sets of specific primers of said modified synthetic DNA fragments specific to said bacteria, and   reagents for implementing a PCR-type DNA amplification reaction.